Studies during the coming year will extend our previous work on the composition of the axonal and dendritic cytoskeletons. We will focus on microtubules (MT), which are composed of tubulin and microtubule-associated proteins (MAPs). Tubulin, which is the major subunit of MT, consists of a-tubulins and B-tubulins, each of which are composed of several isoforms. We will determine whether the tubulins of axonal MT differ from those of cell body + dendritic MT. Explant cultures of rat sympathetic neurons will be used for these experiments because they permit the ready preparation of pure axons and fractions enriched in cell bodies and dendrites. Tubulin will be immunoprecipitated from pure axon and cell body + dendrite-enriched fractions and analyzed by isoelectric focusing. These experiments will show which tubulin isoforms are present in axons as well as cell bodies and dendrites, and also whether any isoforms are uniquely present or greatly enriched in one region of the neuron compared to others. We have previously shown that axonal MT differ from dendritic MT with respect to their MAPs, and have hypothesized that these differences contribute to the well documented differences seen in the spacing between MT of axons compared to dendrites. In vitro experiments will be carried out to test this hypothesis. MT will be reconstituted from pure tubulin plus varying amounts of axonal or dendritic MAPs. The resulting MT will be centrifuged and the packing density of the MT will be quantified biochemically and electron microscopically. The results will reveal whether axon specific MAPs and dendrite specific MAPs have differing effects on the lateral spacing of microtubules under these controlled in vitro conditions. Finally, we will extend our recent work on the phosphorylation of coated membrane proteins of cultured neurons. Coated membranes are involved in a variety of physiological processes including membrane re-cycling following depolarization and stimulation with various hormones. We have recently shown that specific proteins of the coat structure of coated membranes are phosphorylated in intact neurons. Biochemical and immunochemical methods will be used to determine whether the phosphorylation state of these proteins changes in association with depolarization or stimulation with hormones such as nerve growth factor.